Pulmonary edema in patients causes hypoxemic respiratory failure and significant morbidity and mortality. Fluid is reabsorbed from the lungs by the vectorial Na+ movement out of the airspaces, via apically located Na+ channels and basolateral Na,K-ATPases. In the previous cycle of the grant we have reported that upregulation of the alpha1 and beta1subunits of the Na+ pump increase Na,K-ATPase function in alveolar epithelial cells (AEC) in association with increased lung edema clearance. We have also determined that not only the alpha1, but the alpha2 Na,K-ATPase is expressed in AEC and has a similar contributory role as the alpha1 Na,K-ATPase to alveolar fluid clearance. In the current application we will focus on the role and mechanisms of Na,K-ATPase regulation and alveolar fluid reabsorption via four interrelated specific aims: in studies proposed for specific aim 1 we will determine the pathways of endocytosis and degradation of Na,K-ATPase proteins from the alveolar epithelial cell plasma membrane, in specific aim 2, we will conduct studies to determine the role of Rho-Rock pathways as mechanisms of Na,K-ATPase recruitment and regulation in alveolar epithelial cells, in specific aim 3 we will determine whether the intracellular trafficking of Na,K-ATPase during endocytosis from the plasma membrane and recruitment to the plasma membrane are regulated by cytoskeleton-molecular motors in AEC's and in experiments pertaining to specific aim 4 we will study whether delivering constructs containing cDNA for alpha2 Na,K-ATPase to spontaneously breathing mice and/or rats via electroporation, will result in the overexpression of Na,K-ATPase protein and increased alveolar fluid reabsorption. Experiments have been conducted for each of the specific aims and the preliminary data support the feasibility of this application. We reason that the data from the proposed studies will provide important information on the role and regulation of Na,K-ATPase and active Na+ transport in rodent lungs which will help with the design of new strategies to increase lung edema clearance in patients with hypoxemic respiratory failure.